The long-term objectives of this proposal are to (1) investigate the role of somatostatin and epinephrine in negative regulation of insulin gene transcription and translation; (2) determine the mechanisms through which this regulation occurs, and (3) determine whether similar negative regulation is an essential feature of other endogenous inhibitors of insulin secretion. the specific aims and experimental methods to achieve these objectives are: (1) To determine whether the effects of somatostatin and epinephrine to lower insulin mRNA levels in HIT cells are due to decreases in the rate of insulin gene transcription, increases in the rate of insulin mRNA degradation, or both. Gene transcription rates in the presence of somatostatin and epinephrine will be assessed by reporter gene transcription experiments and nuclear run-on assay. Effects on mRNA metabolism will be assessed by determining insulin mRNA half-life by Northern blot analysis in the presence of the transcription inhibitor Actinomycin D. (2) To determine whether the effects of somatostatin and epinephrine to lower intracellular insulin content in HIT cells are due to a decrease in the rate of insulin mRNA translation by measuring the rate of incorporation of labeled amino acids into proinsulin in the presence of somatostatin and epinephrine. (3) To determine whether effects of somatostatin and epinephrine on insulin gene transcription and translation involve either the adenylate cyclase-cAMP second messenger pathway or calcium mobilization by measuring the effects of somatostatin and epinephrine on insulin gene transcription/translation rates in the presence of exogenous cAMP analogues, forskolin or extracellular Ca2+ mobilization by the ionophore ionomycin. (4) To determine whether inhibitory effects of somatostatin and epinephrine on insulin gene transcription and translation are specific to these hormones or whether other inhibitors of insulin secretion whose effects are or are not G-protein mediated have similar effects. This aim will be addressed through experiments described under Aims #1-#3 carried out in the presence of the G-protein mediated inhibitor prostaglandin E2, and the non-G protein mediated inhibitors of insulin secretion diazoxide, verapamil and colchicine. (5) If inhibitory effects of somatostatin and epinephrine on insulin gene transcription are verified, to determine whether specific cis-acting elements in the insulin gene 5'- flanking regulatory region are responsible for these effects by carrying out reporter gene transfection studies in which specific areas of the human insulin gene implicated in cAMP or glucose mediated activation of insulin gene transcription have been selectively mutated or deleted. At the conclusion of these studies, we hope to determine whether endogenous inhibitors of insulin secretion are negative regulators of the beta cell at multiple levels including gene transcription and translation as well as insulin secretion. Such coordinated regulation may be important in glucose homeostasis in physiologic (e.g., exercise, fasting) and pathophysiologic states.